Liver-targeting self-assembled hyaluronic acid-glycyrrhetinic acid micelles enhance hepato-protective effect of silybin after oral administration

Figures & data

Figure 1. (A) Synthetic route of HA-adh-DOCA conjugate and HA-adh-GA conjugate. (B) ¹H NMR spectra of HA (B1), DOCA (B2), GA (B3), HA-adh-DOCA conjugate (B4) and HA-adh-GA conjugate (B5). (C) DSC profiles of HA-adh-DOCA₁₀ conjugate (C1); silybin (C2); physical mixture of HA-adh-DOCA₁₀ conjugate and silybin (C3); and silybin-loaded HA-adh-DOCA₁₀ micelles (C4). (D) TEM image of silybin-loaded HA-adh-DOCA₁₀ micelles (D1) and silybin-loaded HA-adh-GA₂₀ micelles (D2). The magnification bar denotes 100 nm.

Table 1. Characteristics of silybin-loaded HA-adh-DOCA₁₀ micelles and HA-adh-GA₂₀ micelles.

Sample	DS^a	EE (%)^b	DL (wt%)^b	Size^c (nm) (µ2/Г^2,d)	Zeta potential (mV)
HA-adh-DOCA10^e	10.1	92.1 ± 2.6	20.3 ± 0.5	128.1 ± 6.5 (0.119)	−28.9 ± 3.1
HA-adh-GA20^e	20.3	93.4 ± 2.9	20.6 ± 0.6	121.6 ± 5.8 (0.114)	−27.7 ± 2.2

Data are represented as mean ± S.D. (n = 3).

^aDegree of substitution (DS) of deoxycholic acid and glycyrrhetinic acid.

^bEE and DL are short for entrapment efficiency and drug-loading, respectively.

^cMean diameters of micelles.

^dPolydispersity index of micelles size.

^eThe number represents the DS of deoxycholic acid and glycyrrhetinic acid.

Table 2. Stability studies of silybin-loaded HA-adh-DOCA₁₀ micelles and HA-adh-GA₂₀ micelles in simulated gastrointestinal fluids and PBS (pH 7.4).

	Particle size (nm)		Polydispersity index		Zeta potential (mV)
Medium	Initial	Final	Initial	Final	Initial	Final
Silybin-loaded HA-adh-DOCA10 micelles
SGF pH 1.2 2 h		152.8 ± 4.7		0.137 ± 0.043		−0.3 ± 0.1
SIF pH 6.8 6 h	124.5 ± 4.2	123.6 ± 3.8	0.117 ± 0.012	0.121 ± 0.016	−27.1 ± 2.1	−26.6 ± 2.2
PBS pH 7.4 6 h		124.1 ± 4.5		0.119 ± 0.018		−27.2 ± 1.8
Silybin-loaded HA-adh-GA20 micelles
SGF pH 1.2 2 h		153.2 ± 3.3		0.142 ± 0.046		−0.6 ± 0.2
SIF pH 6.8 6 h	123.2 ± 3.8	128.7 ± 3.1	0.111 ± 0.011	0.115 ± 0.019	−27.5 ± 1.8	−27.3 ± 1.7
PBS pH 7.4 6 h		128.3 ± 2.9		0.118 ± 0.018		−27.8 ± 1.6

Data are represented as mean ± S.D. (n = 3).

Figure 2. Cumulative released amount of silybin as a function of time in SGF (pH = 1.2, 0–2 h) and SIF (pH = 6.8, 2–48 h) and in PBS (pH = 7.4, 0–48 h). Data are represented as the mean ± S.D. (n = 3).

Figure 3. (A) Effective permeability (P_eff) of silybin-loaded HA-adh-DOCA₁₀ and HA-adh-GA₂₀ micelles using single-pass intestinal perfusion. (B) Influence of different drug concentrations on the P_eff of silybin across rat ileum segment. (C) Plasma concentration-time profiles of silybin after oral administration of silybin suspension, silybin-loaded HA-adh-DOCA₁₀ micelles and silybin-loaded HA-adh-GA₂₀ micelles at a dose of 30 mg/kg to Sprague-Dawley rats. Data are represented as mean ± S.D. (n = 6).

Table 3. Pharmacokinetic parameters of silybin formulations after a single oral dose of 30 mg/kg to Sprague-Dawley rats.

Groups	Cmax (µg/mL)	tmax (h)	AUC0–t (µg h/mL)	MRT (h)
Silybin suspension	0.418 ± 0.051	0.17	1.011 ± 0.203	3.615 ± 0.317
HA-adh-DOCA10	0.885 ± 0.145**	1.0	4.429 ± 0.682**	4.927 ± 0.612*
HA-adh-GA20	0.879 ± 0.132**	0.5	4.074 ± 0.657**	4.746 ± 0.563*

Data are represented as mean ± S.D. (n = 6).

*p < 0.05 and **p < 0.01 versus silybin suspension formulation group.

Figure 4. In vivo imaging of ICR mice administrated with DiR-loaded micelles. (A) Images were taken after oral administration of HA-adh-DOCA₁₀ and HA-adh-GA₂₀ micelles at 1 h, 6 h and 12 h, respectively. (B) Ex vivo fluorescence images of tissues collected at 12 h post-administration of HA-adh-DOCA₁₀ and HA-adh-GA₂₀ micelles.

Figure 5. (A) Effects of silybin suspension, silybin-loaded HA-adh-DOCA₁₀ micelles and silybin-loaded HA-adh-GA₂₀ micelles on the AST and ALT activity. Each of the data was presented as mean ± SD (n = 10). ***p < 0.001 versus CCl₄ group. #p < 0.05, ##p < 0.01 and ###p < 0.001 versus silybin-loaded HA-adh-GA₂₀ micelles group. (B) Histological analysis of liver slices after treatment of saline (B1, normal control), CCl₄ (B2), silybin suspension formulation (B3), silybin-loaded HA-adh-DOCA₁₀ micelles (B4) and silybin-loaded HA-adh-GA₂₀ micelles (B5) by H & E staining. All the images were captured by the Nikon microscope at a 200× magnification of the original images.